The present invention concerns novel HER2-transgenic non-human mammals and their use to develop a tumor model to test HER2-directed cancer therapies. The invention further concerns anticancer agents identified in this model, and their use in the treatment of cancer.
Members of the ErbB family of receptor tyrosine kinases are important mediators of cell growth, differentiation and survival. The receptor family includes four distinct members, including epidermal growth factor receptor (EGFR or ErbB 1), HER2 (ErbB2 or p185neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).
p185neu, was originally identified as the product of the transforming gene from neuroblastomas of chemically treated rats. The activated form of the neu proto-oncogene results from a point mutation (valine to glutamic acid) in the transmembrane region of the encoded protein. Amplification of the human homolog of neu is observed in breast and ovarian cancers and correlates with a poor prognosis (Slamon et al., Science, 235:177-182 (1987); Slamon et al., Science, 244:707-712 (1989); and U.S. Pat No. 4,968,603). To date, no point mutation analogous to that in the neu proto-oncogene has been reported for human tumors. Overexpression of ErbB2 (frequently but not uniformly due to gene amplification) has also been observed in other carcinomas including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas and bladder. See, among others, King et al., Science, 229:974 (1985); Yokota et al., Lancet, 1:765-767 (1986); Fukushigi et al., Mol Cell Biol., 6:955-958 (1986); Geurin et al., Oncogene Res., 3:21-31 (1988); Cohen et al., Oncogene, 4:81-88 (1989); Yonemura et al., Cancer Res., 51:1034 (1991); Borst et al., Gynecol. Oncol., 38:364 (1990); Weiner et al., Cancer Res., 50:421-425 (1990); Kern et al., Cancer Res., 50:5184 (1990); Park et al., Cancer Res., 49:6605 (1989); Zhau et al., Mol. Carcinog., 3:354-357 (1990); Aasland et al. Br. J. Cancer 57:358-363 (1988); Williams et al. Pathobiology 59:46-52 (1991); and McCann et al., Cancer, 65:88-92 (1990). ErbB2 may be overexpressed in prostate cancer (Gu et al. Cancer Lett. 99:185-9 (1996); Ross et al. Hum. Pathol. 28:827-33 (1997); Ross et al. Cancer 79:2162-70 (1997); and Sadasivan et al. J. Urol. 150:126-31 (1993)).
Antibodies directed against the rat p185neu and human ErbB2 protein products have been described. Drebin and colleagues have raised antibodies against the rat neu gene product, p185neu. See, for example, Drebin et al., Cell 41:695-706 (1985); Myers et al., Meth. Enzym. 198:277-290 (1991); and W094/22478. Drebin et al. Oncogene 2:273-277 (1988) report that mixtures of antibodies reactive with two distinct regions of p185neu result in synergistic anti-tumor effects on neu-transformed NIH-3T3 cells implanted into nude mice. See also U.S. Pat. 5,824,311 issued Oct. 20, 1998.
Other anti-ErbB2 antibodies with various properties have been described in Tagliabue et al. Int. J. Cancer 47:933-937 (1991); McKenzie et al. Oncogene 4:543-548 (1989); Maier et al. Cancer Res. 51:5361-5369 (1991); Bacus et al. Molecular Carcinogenesis 3:350-362 (1990); Stancovski et al. PNAS (USA) 88:8691-8695 (1991); Bacus et al. Cancer Research 52:2580-2589 (1992); Xu et al. Int. J. Cancer 53:401-408 (1993); W094/00136; Kasprzyk et al. Cancer Research 52:2771-2776 (1992);Hancock et al. Cancer Res. 51:4575-4580 (1991); Shawver et al. Cancer Res. 54:1367-1373 (1994); Arteaga et al. Cancer Res. 54:3758-3765 (1994); Harwerth et al. J. Biol. Chem. 267:15160-15167 (1992); U.S. Pat. No. 5,783,186; and Klapper et al. Oncogene 14:2099-2109 (1997).
Hudziak et al., Mol. Cell. Biol. 9(3): 1165-1172 (1989) describe the generation of a panel of anti-ErbB2 antibodies which were characterized using the human breast tumor cell line SK-BR-3. Relative cell proliferation of the SK-BR-3 cells following exposure to the antibodies was determined by crystal violet staining of the monolayers after 72 hours. Using this assay, maximum inhibition was obtained with the antibody called 4D5 which inhibited cellular proliferation by 56%. Other antibodies in the panel reduced cellular proliferation to a lesser extent in this assay. The antibody 4D5 was further found to sensitize ErbB2-overexpressing breast tumor cell lines to the cytotoxic effects of TNF-xcex1. See also U.S. Pat. No. 5,677,171 issued Oct. 14, 1997. The anti-ErbB2 antibodies discussed in Hudziak et al. are further characterized in Fendly et al. Cancer Research 50:1550-1558 (1990); Kotts et al. In Vitro 26(3):59A (1990); Sarup et al. Growth Regulation 1:72-82 (1991); Shepard et al. J. Clin. Immunol. 11(3):117-127 (1991); Kumar et al. Mol. Cell. Biol. 11(2):979-986 (1991); Lewis et al. Cancer Immunol. Immunother. 37:255-263 (1993); Pietras et al. Oncogene 9:1829-1838 (1994); Vitetta et al. Cancer Research 54:5301-5309 (1994); Sliwkowski et al. J. Biol. Chem. 269(20):14661-14665 (1994); Scott et al. J. Biol. Chem. 266:14300-5 (1991); D""souza et al. Proc. Natl. Acad. Sci. 91:7202-7206 (1994); Lewis et al. Cancer Research 56:1457-1465 (1996); and Schaefer et al. Oncogene 15:1385-1394 (1997).
The murine monoclonal anti-HER2 antibody inhibits the growth of breast cancer cell lines that overexpress HER2 at the 2+ and 3+ level, but has no activity on cells that express lower levels of HER2 (Lewis et al., Cancer Immunol. Immunother. [1993]). Based on this observation, antibody 4D5 was humanized (Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285-4289 [1992]). The humanized version designated HERCEPTIN(copyright) (huMAb4D5-8, rhuMAb HER2, U.S. Pat. No. 5,821,337) was tested in breast cancer patients whose tumors overexpress HER2 but who had progressed after conventional chemotherapy (Cobleigh et al., J. Clin. Oncol. 17: 2639-2648 [1999]). Most patients in this trial expressed HER2 at the 3+ level, though a fraction was 2+ tumors. Remarkably, HERCEPTIN(copyright) induced clinical responses in 15% of patients (complete responses in 4% of patients, and partial responses in 11%) and the median duration of those responses was 9.1 months. HERCEPTIN(copyright) received marketing approval from the Food and Drug Administration Sep. 25, 1998 for the treatment of patients with metastatic breast cancer whose tumors overexpress the ErbB2 protein.
Although HERCEPTINxc2x0 is a breakthrough in treating patients with ErbB2-overexpressing breast cancers that have received extensive prior anti-cancer therapy, generally approximately 85% of the patients in this population fail to respond or respond only poorly to HERCEPTIN(copyright) treatment, and in the clinical trial preceding market approval, the median time to disease progression in all treated patients was only 3.1 months.
Therefore, there is a significant clinical need for developing further HER2-directed cancer therapies for those patients with HER2-overexpressing tumors or other diseases associated with HER2 expression that do not respond or respond poorly to HERCEPTIN(copyright) treatment.
The present invention is based on the development of a novel HER2-transgenic mouse tumor model that expresses HER2 at high levels, comparable to those in HER2-positive human patients, but that responds poorly to HERCEPTIN(copyright).
In one aspect, the invention concerns a transgenic non-human mammal that produces in its mammary gland cells detectable levels of a native human HER2 protein or a fragment thereof, wherein said transgenic mammal has stably integrated into its genome a nucleic acid sequence encoding a native human HER2 protein or a fragment thereof having the biological activity of native human HER2, operably linked to transcriptional regulatory sequences directing its expression to the mammary gland, and develops a mammary tumor not responding or poorly responding to anti-HER2 antibody treatment. The transcriptional regulatory sequences preferably comprise a mammary gland specific promoter, such as the glucocorticoid-inducible promoter present in the mouse mammary tumor virus long terminal repeat (MMTV LTR). The non-human transgenic mammal preferably overexpresses the HER2 protein, more preferably, it expresses native human HER2 protein in at least about 500,000 copies per cell, even more preferably in at least about 2,000,000 copies per cell. Without limitation, the non-human transgenic mammal may, for example, be mouse, rat, rabbit, pig, sheep, goat or cattle. In a particular embodiment, the HER2-transgenic mammal develops a tumor that does not respond or responds poorly to treatment by a humanized version of the murine anti-HER2 antibody 4D5, such as HERCEPTIN(copyright).
In another aspect, the invention concerns a non-human animal model for HER2-expressing tumors comprising a non-human mammal bearing a tumor overexpressing HER2 and not responding or poorly responding to anti-HER2 antibody treatment. The tumor preferably expresses a human HER2 protein in at least about 500,000 copies per cell, more preferably in at least about 2,000,000 copies per cell. In a preferred embodiment, the tumor has been transplanted from a transgenic non-human mammal which has stably integrated into its genome a nucleic acid sequence encoding a native human HER2 protein or a fragment thereof having the biological activity of native human HER2, operably linked to transcriptional regulatory sequences directing its expression to the mammary gland.
In yet another aspect, the invention concerns a transgene construct comprising nucleic acid encoding a native human HER2 protein or a fragment thereof, under the control of transcriptional regulatory sequences directing its expression to the mammary gland. The transgene construct preferably comprises a mammary gland specific promoter, such as an MMTV-LTR promoter.
In a further aspect, the invention concerns a stable cell line established from a HER2-transgenic non-human mammal that produces in its mammary gland cells detectable levels of a native human HER2 protein or a fragment thereof, wherein such transgenic mammal has stably integrated into its genome a nucleic acid sequence encoding a native human HER2 protein or a fragment thereof having the biological activity of native human HER2, operably linked to transcriptional regulatory sequences directing its expression to the mammary gland, and develops a mammary tumor not responding or poorly responding to anti-HER2 antibody treatmement. In one embodiment the stable cell line is a breast cancer cell line.
In one embodiment the stable breast cancer cell line is selected from the group consisting of HER-32, HER-3081-3-4, HER-3081-3-19, HER-3081-3-33, HER-3081-3-12C and HER-3080-5L1. In another embodiment the stable breast cancer cell line is selected from the group consisting of the stable cell lines HER-5, HER-3081-3-1 and HER-3081-3-10 deposited with ATCC on Feb. 28, 2001 under accession numbers PTA-3135, PTA-3133 and PTA-3134,
In another aspect, the invention concerns a method of screening drug candidates for the treatment of a disease or disorder characterized by the overexpression of HER2 comprising (a) transplanting cells from a stable breast cancer cell line into a non-human animal, (b) administering a drug candidate to the non-human animal and (c) determining the ability of the candidate to inhibit the formation of tumors from the transplanted cell line.
The invention also concerns a method of screening drug candidates for the treatment of a disease or disorder characterized by the overexpression of HER2 comprising (a) contacting cells from a stable breast cancer cell line with a drug candidate and (b) evaluating the ability of the drug candidate to inhibit the growth of the stable cell line.
In another aspect the invention concerns a method of screening drug candidates for the treatment of a disease or disorder characterized by the overexpression of HER2 comprising (a) contacting cells from a stable breast cancer cell line with a drug candidate and (b) evaluating the ability of the drug candidate to block ligand activation of HER2. In one embodiment the ability of the drug candidate to block heregulin binding is evaluated. In another embodiment the ability of the drug candidate to block ligand-stimulated tyrosine phosphorylation is evaluated.
In yet another aspect, the invention concerns a method of screening drug candidates for the treatment of a disease or disorder characterized by the overexpression of HER2 comprising (a) contacting cells from a stable breast cancer cell line with a drug candidate and (b) evaluating the ability of the drug candidate to induce cell death. In one embodiment the ability of the drug candidate to induce apoptosis is evaluated.
In a still further aspect, the invention concerns a method of screening drug candidates for the treatment of a disease or disorder characterized by the overexpression of HER2 comprising (a) administering a drug candidate to a transgenic non-human mammal that overexpresses in its mammary gland cells a native human HER2 protein or a fragment thereof, wherein such transgenic mammal has stably integrated into its genome a nucleic acid sequence encoding a native human HER2 protein or a fragment thereof having the biological activity of native human HER2, operably linked to transcriptional regulatory sequences directing its expression to the mammary gland, and develops a mammary tumor not responding or poorly responding to anti-HER2 antibody treatment, or to a non-human mammal bearing a tumor transplanted from said transgenic non-human mammal; and (b) evaluating the effect of the drug candidate on the target disease or disorder. Without limitations, the disease or disorder may be a HER2-overexpressing cancer, such as breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic and bladder cancer. The cancer preferably is breast cancer which expressed HER2 in at least about 500,000 copies per cell, more preferably at least about 2,000,000 copies per cell. Drug candidates may, for example, be evaluated for their ability to induce cell death and/or apoptosis, using assay methods well known in the art and described hereinafter. The drug candidates may, for example, be antibodies or other polypeptides, antibody conjugates, peptides, or organic or inorganic small molecules.
The invention further concerns anti-cancer molecules identifiable in the animal models and other assays of the present invention, and compositions comprising such molecules in admixture with a pharmaceutically acceptable carrier.
FIGS. 1A-GG shows the nucleotide sequence of a HER2 transgene plasmid construct (SEQ ID NO: 1) directing the expression of native human HER2 in the mammary gland of a transgenic mouse. The reverse strand is also depicted (SEQ ID NO: 4). The figure includes the nucleotide sequence of HER2 cDNA insert (SEQ ID NO: 2) as well as the deduced amino acid sequence of HER2 (SEQ ID NO: 3).
FIGS. 2A-B The amount of HER2 ECD shed into serum increases following transplant (FIG. 2A) and is proportional to tumor weight (FIG. 2B).
FIG. 3 Effect of HERCEPTIN(copyright)-maytansinoid on HER2-transgenic tumors. Two mm3 pieces of MMTV-HER2-transgenic tumors were transplanted into the mammary fat pad of FVB mice. When tumors reached 250 mm3, groups of 8 mice were injected i.v. on 5 consecutive days with a HERCEPTIN(copyright)-maytansinoid conjugate. Two other groups of mice were treated IP twice per week with 10 mg/kg of either HERCEPTIN(copyright) or RITUXAN(copyright).
FIG. 4 Tumor cells originating from Founder 5 show binding to cy3-HERCEPTIN(copyright) and to anti-tyrosine phosphorylated HER2 antibody. Antibodies were injected intravenously into transgenic mice and the next day tumors were collected and sectioned. Antibody binding was visualized by fluoresence microscopy.
FIG. 5 Weekly administration of 4D5 can significantly reduce the formation of mammary tumors in HER2 transgenic carrier female mice.
FIG. 6 Structure of a HERCEPTIN(copyright)-maytansinoid conjugate.
FIG. 7 Effect of different HERCEPTIN(copyright)-maytansinoid (Herceptin-DM1) dosing regimens on HER2-transgenic tumors. Mice with 100 mm3 tumors were injected i.v. with HERCEPTIN(copyright)-DM1) or RITUXAN(copyright)-maytansinoid (RITUXAN(copyright)-DM1) at doses of 100 or 300 xcexcg DM1/kg twice a week or 300 xcexcg DM1/kg once a week. All animals received five doses.
FIG. 8 Comparison of the most effective observed dose of HERCEPTIN(copyright)-DM1and RITUXAN(copyright)-DM1. Mice with 100 mm3 tumors were treated with 300 xcexcg DM1/kg of either HERCEPTIN(copyright)-DM1 or RITUXAN(copyright)-DM1 once a week for 5 weeks.
FIG. 9 depicts the morphology of several of the cell lines developed from HER2 transgenic mice.
FIG. 10 shows expression of human HER2 on transgenic cell lines as determined by the binding of fluorescently labeled anti-HER2 antibodies 4D5, 2C4 and 7C2.
FIG. 11 shows that heregulin and EGF are mitogens only for cell line HER-5 and not for cell lines HER-3081-3-4, HER-3081-3-10 or HER-3081-3-19.
FIG. 12 is a summary of the growth response of HER2 transgenic cell lines to anti-HER2 antibodies in the presence or absence of mitogens.
FIG. 13 shows the phosphorylation state of the human HER2 receptor in HER2 transgenic cell lines in both the presence and absence of mitogens.
FIG. 14 shows the anchorage independent growth of HER2 transgenic cells after treatment with anti-HER2 antibodies.
FIGS. 15A-B shows that a number of the transgenic HER2 cell lines, including one from Founder 5 (FIG. 15A) and several from Founder 3081-3 (FIG. 15B), are capable of forming tumors in immunocompetant mice.
FIG. 16 shows the effect of taxol and anti-HER2 monoclonal antibodies 4D5 and 7C2 on the growth of tumors from the HER2 transgenic cell line HER-3081-3-10.